Single Molecule Methods for studying CRISPR Cas9-Induced DNA Unwinding

Human DNA2 (hDNA2) contains both a helicase and a nuclease domain within the same polypeptide. The nuclease of hDNA2 is involved in a variety of DNA metabolic processes. Little is known about the role of the hDNA2 helicase. Using bulk and single-molecule approaches, we show that hDNA2 is a processive helicase capable of unwinding kilobases of dsDNA in length. The nuclease activity prevents the engagement of the helicase by competing for the same substrate, hence prominent DNA unwinding by hDNA2 alone can only be observed using the nuclease-deficient variant. We show that the helicase of hDNA2 functionally integrates with BLM or WRN helicases to promote dsDNA degradation by forming a heterodimeric molecular machine. This collectively suggests that the hDNA2 motor promotes the enzyme's capacity to degrade dsDNA in conjunction with BLM or WRN and thus promote the repair of broken DNA.

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Mechanisms of improved specificity of engineered Cas9s revealed by single molecule analysis

10.1101/192724 ◽

2017 ◽

Cited By ~ 2

Author(s):

Digvijay Singh ◽

Yanbo Wang ◽

John Mallon ◽

Olivia Yang ◽

Jingyi Fei ◽

...

Keyword(s):

Single Molecule ◽

Dna Unwinding ◽

Cleavage Reaction ◽

Cleavage Rate ◽

Guide Rna ◽

Slowing Down ◽

Rna Targets ◽

Cleavage Reactions ◽

Additional Base ◽

Single Molecule Analysis

ABSTRACTIn microbes, CRISPR-Cas systems provide adaptive immunity against invading genetic elements. Cas9 in complex with a guide-RNA targets complementary DNA for cleavage and has been repurposed for wide-ranging biological applications. New Cas9s have been engineered (eCas9 and Cas9-HF1) to improve specificity, but how they help reduce off-target cleavage is not known. Here, we developed single molecule DNA unwinding assay to show that sequence mismatches affect cleavage reactions through rebalancing the internal unwinding/rewinding equilibrium. Increasing PAM-distal mismatches facilitate rewinding, and the associated cleavage impairment shows that cleavage proceeds from the unwound state. Engineered Cas9s depopulate the unwound state more readily upon mismatch detection. Intrinsic cleavage rate is much lower for engineered Cas9s, preventing cleavage from transiently unwound off-targets. DNA interrogation experiments showed that engineered Cas9s require about one additional base pair match for stable binding, freeing them from sites that would otherwise sequester them. Therefore, engineered Cas9s achieve their improved specificity (1) by inhibiting stable DNA binding to partially matching sequences, (2) by making DNA unwinding more sensitive to mismatches, and (3) by slowing down intrinsic cleavage reaction.

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Kinetic and structural mechanism for DNA unwinding by a non-hexameric helicase

Nature Communications ◽

10.1038/s41467-021-27304-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Sean P. Carney ◽

Wen Ma ◽

Kevin D. Whitley ◽

Haifeng Jia ◽

Timothy M. Lohman ◽

...

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Atp Hydrolysis ◽

Variable Number ◽

Structural Basis ◽

Variable Step Size ◽

Dna Unwinding ◽

Step Size ◽

Structural Mechanism ◽

Dynamics Simulations

AbstractUvrD, a model for non-hexameric Superfamily 1 helicases, utilizes ATP hydrolysis to translocate stepwise along single-stranded DNA and unwind the duplex. Previous estimates of its step size have been indirect, and a consensus on its stepping mechanism is lacking. To dissect the mechanism underlying DNA unwinding, we use optical tweezers to measure directly the stepping behavior of UvrD as it processes a DNA hairpin and show that UvrD exhibits a variable step size averaging ~3 base pairs. Analyzing stepping kinetics across ATP reveals the type and number of catalytic events that occur with different step sizes. These single-molecule data reveal a mechanism in which UvrD moves one base pair at a time but sequesters the nascent single strands, releasing them non-uniformly after a variable number of catalytic cycles. Molecular dynamics simulations point to a structural basis for this behavior, identifying the protein-DNA interactions responsible for strand sequestration. Based on structural and sequence alignment data, we propose that this stepping mechanism may be conserved among other non-hexameric helicases.

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A single-molecule mechanical assay to study DNA replication coupled to DNA unwinding

EMC 2008 14th European Microscopy Congress 1–5 September 2008, Aachen, Germany ◽

10.1007/978-3-540-85228-5_112 ◽

2008 ◽

pp. 223-224

Author(s):

B. Ibarra ◽

J. A. Morin ◽

R. Arias ◽

S. Hormeño ◽

M. Salas ◽

...

Keyword(s):

Dna Replication ◽

Single Molecule ◽

Dna Unwinding

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Single-molecule visualization of RecQ helicase reveals DNA melting, nucleation, and assembly are required for processive DNA unwinding

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1518028112 ◽

2015 ◽

Vol 112 (50) ◽

pp. E6852-E6861 ◽

Cited By ~ 23

Author(s):

Behzad Rad ◽

Anthony L. Forget ◽

Ronald J. Baskin ◽

Stephen C. Kowalczykowski

Keyword(s):

Single Molecule ◽

Fluorescent Sensor ◽

Holliday Junctions ◽

Dna Unwinding ◽

Recq Helicase ◽

Dna Breaks ◽

Dna Helicases ◽

Double Stranded Dna ◽

Functional Forms ◽

And Migration

DNA helicases are motor proteins that unwind double-stranded DNA (dsDNA) to reveal single-stranded DNA (ssDNA) needed for many biological processes. The RecQ helicase is involved in repairing damage caused by DNA breaks and stalled replication forks via homologous recombination. Here, the helicase activity of RecQ was visualized on single molecules of DNA using a fluorescent sensor that directly detects ssDNA. By monitoring the formation and progression of individual unwinding forks, we observed that both the frequency of initiation and the rate of unwinding are highly dependent on RecQ concentration. We establish that unwinding forks can initiate internally by melting dsDNA and can proceed in both directions at up to 40–60 bp/s. The findings suggest that initiation requires a RecQ dimer, and that continued processive unwinding of several kilobases involves multiple monomers at the DNA unwinding fork. We propose a distinctive model wherein RecQ melts dsDNA internally to initiate unwinding and subsequently assembles at the fork into a distribution of multimeric species, each encompassing a broad distribution of rates, to unwind DNA. These studies define the species that promote resection of DNA, proofreading of homologous pairing, and migration of Holliday junctions, and they suggest that various functional forms of RecQ can be assembled that unwind at rates tailored to the diverse biological functions of RecQ helicase.

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Single-molecule studies reveal reciprocating of WRN helicase core along ssDNA during DNA unwinding

Scientific Reports ◽

10.1038/srep43954 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 11

Author(s):

Wen-Qiang Wu ◽

Xi-Miao Hou ◽

Bo Zhang ◽

Philippe Fossé ◽

Brigitte René ◽

...

Keyword(s):

Single Molecule ◽

Dna Unwinding ◽

Single Molecule Studies ◽

Wrn Helicase

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Bulk and single-molecule analysis of a novel DNA2-like helicase-nuclease reveals a single-stranded DNA looping motor

10.1101/851329 ◽

2019 ◽

Author(s):

O.J. Wilkinson ◽

C. Carrasco ◽

C. Aicart-Ramos ◽

F. Moreno-Herrero ◽

M.S. Dillingham

Keyword(s):

Single Molecule ◽

Nuclease Activity ◽

Biochemical Properties ◽

Dna Helicase ◽

Dna Looping ◽

Dna Unwinding ◽

Ssdna Binding ◽

Dna Replication And Repair ◽

Essential Enzyme ◽

Single Molecule Analysis

ABSTRACTDNA2 is an essential enzyme involved in DNA replication and repair in eukaryotes. In a search for homologues of this protein, we identified and characterised Geobacillus stearothermophilus Bad, a novel bacterial DNA helicase-nuclease with similarity to human DNA2. We show that Bad contains an Fe-S cluster and identify four cysteine residues that are likely to co-ordinate the cluster by analogy to DNA2. The purified enzyme specifically recognises ss-dsDNA junctions and possesses ssDNA-dependent ATPase, ssDNA binding, ssDNA endonuclease, 5’ to 3’ ssDNA translocase and 5’ to 3’ helicase activity. Single molecule analysis reveals that Bad is a highly processive DNA motor capable of moving along DNA for distances of more than 4 kbp at a rate of ∼200 base pairs per second at room temperature. Interestingly, as reported for the homologous human and yeast DNA2 proteins, the DNA unwinding activity of Bad is cryptic and can be unmasked by inactivating the intrinsic nuclease activity. Strikingly, our experiments also show that the enzyme loops DNA while translocating, which is an emerging feature of highly processive DNA unwinding enzymes. The bacterial Bad enzymes will provide an excellent model system for understanding the biochemical properties of DNA2-like helicase-nucleases and DNA looping motor proteins in general.

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Hexameric helicase G40P unwinds DNA in single base pair steps

eLife ◽

10.7554/elife.42001 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 8

Author(s):

Michael Schlierf ◽

Ganggang Wang ◽

Xiaojiang S Chen ◽

Taekjip Ha

Keyword(s):

Single Molecule ◽

Base Pair ◽

Atp Hydrolysis ◽

Thermal Fluctuation ◽

Dna Unwinding ◽

Single Molecule Fluorescence ◽

Step Size ◽

Structural Investigations ◽

Single Base ◽

Single Base Pair

Most replicative helicases are hexameric, ring-shaped motor proteins that translocate on and unwind DNA. Despite extensive biochemical and structural investigations, how their translocation activity is utilized chemo-mechanically in DNA unwinding is poorly understood. We examined DNA unwinding by G40P, a DnaB-family helicase, using a single-molecule fluorescence assay with a single base pair resolution. The high-resolution assay revealed that G40P by itself is a very weak helicase that stalls at barriers as small as a single GC base pair and unwinds DNA with the step size of a single base pair. Binding of a single ATPγS could stall unwinding, demonstrating highly coordinated ATP hydrolysis between six identical subunits. We observed frequent slippage of the helicase, which is fully suppressed by the primase DnaG. We anticipate that these findings allow a better understanding on the fine balance of thermal fluctuation activation and energy derived from hydrolysis.

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Human HELB is a processive motor protein which catalyses RPA clearance from single-stranded DNA

10.1101/2021.05.27.445972 ◽

2021 ◽

Author(s):

Silvia Hormeno ◽

Oliver J Wilkinson ◽

Clara Aicart-Ramos ◽

Sahiti Kuppa ◽

Edwin Antony ◽

...

Keyword(s):

Dna Replication ◽

Direct Observation ◽

Single Molecule ◽

Atp Hydrolysis ◽

Motor Protein ◽

Dna Unwinding ◽

Helicase Activity ◽

Dna Loops ◽

Single Stranded Dna ◽

A Site

Human HELB is a poorly-characterised helicase suggested to play both positive and negative regulatory roles in DNA replication and recombination. In this work, we used bulk and single molecule approaches to characterise the biochemical activities of HELB protein with a particular focus on its interactions with RPA and RPA-ssDNA filaments. HELB is a monomeric protein which binds tightly to ssDNA with a site size of ~20 nucleotides. It couples ATP hydrolysis to translocation along ssDNA in the 5′-to-3′ direction accompanied by the formation of DNA loops and with an efficiency of 1 ATP per base. HELB also displays classical helicase activity but this is very weak in the absence of an assisting force. HELB binds specifically to human RPA which enhances its ATPase and ssDNA translocase activities but inhibits DNA unwinding. Direct observation of HELB on RPA nucleoprotein filaments shows that translocating HELB concomitantly clears RPA from single-stranded DNA.

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Repetitive DNA reeling by the Cascade-Cas3 complex in nucleotide unwinding steps

10.1101/207886 ◽

2017 ◽

Cited By ~ 1

Author(s):

Luuk Loeff ◽

Stan J.J. Brouns ◽

Chirlmin Joo

Keyword(s):

Single Molecule ◽

Adaptive Immune System ◽

Dna Unwinding ◽

Type I ◽

Base Pairs ◽

Spatiotemporal Resolution ◽

Adaptive Immune ◽

Single Molecule Fret ◽

Target Dna ◽

Loaded Mechanism

CRISPR-Cas loci provide an RNA-guided adaptive immune system against invading genetic elements. Interference in type I systems relies on the RNA-guided surveillance complex Cascade for target DNA recognition and the trans-acting Cas3 helicase/nuclease protein for target degradation. Even though the biochemistry of CRISPR interference has been largely covered, the biophysics of DNA unwinding and coupling of the helicase and nuclease domains of Cas3 remains elusive. Here we employed single-molecule FRET to probe the helicase activity with a high spatiotemporal resolution. We show that Cas3 remains tightly associated with the target-bound Cascade complex while reeling in the target DNA using a spring-loaded mechanism. This spring-loaded reeling occurs in distinct bursts of three base pairs, that each underlie three successive 1-nt unwinding events. Reeling is highly repetitive, compensating for inefficient nicking activity of the nuclease domain. Our study reveals that the discontinuous helicase properties of Cas3 and its tight interaction with Cascade ensure well controlled degradation of target DNA only.

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